The identification of the biological pathway of action of a drug or drug candidate is a problem of great commercial and human importance. Although the primary molecular target of and cellular pathways affected by a drug are often known or suspected because the drug was originally selected by a specific drug screen, it is important to verify its action on such a primary pathway and to quantify its action along other secondary pathways which may be harmful, or may be beneficial, often in unsuspected ways. In other cases, the primary pathways of drug action are unknown, and these must be determined.
This information is important in many areas of practical research, such as, for example, drug discovery, which is a process by which bioactive compounds are identified and preliminarily characterized. Drug discovery is a critical step in the development of treatments for human diseases. Two approaches presently dominate the search for new drugs. The first begins with a screen for compounds that have a desired effect on a cell (e.g., induction of apoptosis), or organism (e.g., inhibition of angiogenesis) as measured in a specific biological assay. Compounds with the desired activity may then be modified to increase potency, stability, or other properties, and the modified compounds retested in the assay. Thus, a compound that acts as an inhibitor of angiogenesis when tested in a mouse tumor model may be identified, and structurally related compounds synthesized and tested in the same assay. One limitation of this approach is that, often, the mechanisms of action, such as the molecular target(s) and cellular pathways affected by the compound, are unknown, and cannot be determined by the screen. In addition, the assay may provide little information about the specificity, either in terms of targets or pathways, of the drug's effect. Finally, the number of compounds that can be screened by assaying biological effects on cells or animals is limited by the required experimental efforts.
In contrast, the second approach to drug screening involves testing numerous compounds for a specific effect on a known molecular target, typically a cloned gene sequence or an isolated enzyme or protein. For example, high-throughput assays can be developed in which numerous compounds can be tested for the ability to change the level of transcription from a specific promoter or the binding of identified proteins. Although the use of high-throughput screens is a powerful methodology for identifying drug candidates, it has limitations. A major drawback is that the assay provides little or no information about the effects of a compound at the cellular or organismal level, in particular information concerning the actual cellular pathways affected. These effects must be tested by using the drug in a series of cell biologic and whole animal studies to determine toxicity or side effects in vivo. In fact, analysis of the specificity and toxicity studies of candidate drugs can consume a significant fraction of the drug development process (see, e.g., Oliff et al., 1997, "Molecular Targets for Drug Development," in DeVita et al. Cancer: Principles & Practice of Oncology 5th Ed. 1997 Lippincott-Raven Publishers, Philadelphia).
Several gene expression assays are now becoming practicable for quantitating the drug effect on a large fraction of the genes and proteins in a cell culture (see, e.g., Schena et al, 1995, Quantitative monitoring of gene expression patterns with a complementary DNA micro-array, Science 270:467-470; Lockhort et al., 1996, Expression monitoring by hybridization to high-density oligonucleotide arrays, Nature Biotechnology 14:1675-1680; Blanchard et al., 1996, Sequence to array: Probing the genome's secrets, Nature Biotechnology 14, 1649; 1996, U.S. Pat. No. 5,569,588, issued Oct. 29, 1996 to Ashby et al. entitled "Methods for Drug Screening"). Raw data from these gene expression assays are often difficult to coherently interpret. Such measurement technologies typically return numerous genes with altered expression in response to a drug, typically 50-100, possibly up to 1,000 or as few as 10. In the typical case, without more analysis, it is not possible to discern cause and effect from such data alone. The fact that one or a few genes among many has an altered expression in a pair of related biological states yields little or no insight into what caused this change and what the effects of this change are. These data in themselves do not inform an investigator about the pathways affected or mechanism of action. They do not indicate which effects result from effects on a primary pathway versus which effects are the result of other secondary pathways affected by the drug. Knowledge of all these affected pathways individually is useful in understanding efficacy, side-effects, toxicities, possible failures of efficacy, activation of metabolic responses, and so forth. Further, identification of all pathways of drug action can lead to discovery of alternate pathways suitable to achieve the original therapeutic purpose.
Without effective methods of analysis, one is left to ad hoc further experimentation to interpret such gene expression results in terms of biological pathways and mechanisms. Systematic procedures for guiding the interpretation of such data and such further experimentation, at least in the case of drug target screening, are needed.
Thus, there is a need for improved (e.g., faster and less expensive) methods for characterizing drug activities, and cellular pathways affected by drugs based on effective interpretation of such data as gene expression data. The present invention provides methods for rapidly identifying the molecular targets and pathways affected by candidate drugs and for characterizing their specificity. It further provides methods based on measurement methodologies other than gene expression analysis.